Cyclin D1 (PRAD1) has emerged as a major oncogene involved in a variety of human tumors including up to 50 percent of breast cancers. Numerous studies, generally employing in vitro systems, have led to the widely accepted paradigm that cyclin D1 functions in a biochemical pathway with cyclin- dependent kinase (cdk) 4 or 6, which it activates, the pl6 inhibitor of cdk4/6, and the retinoblastoma oncosuppressor protein pRB, thought to be the primary substrate of cyclin D1- activated cdk. These molecules have tremendous significance to cancer, being targeted by mutation or regulatory derangements in most or perhaps even all cancers. Current concepts of cyclin D1 function, however, may not fully reflect its true role when overexpressed in the complex in vivo process of tumorigenesis. Recent data, in fact, indicate that cyclin D1 may contribute to oncogenesis through mechanisms other than, or in addition to, its role in the cdk4/6 - pRB pathway, for example by activating the estrogen receptor in breast cancer cells. Our mammary cancer- prone transgenic mice with targeted overexpression of cyclin D1 constitute an especially relevant system in which to test such hypotheses. The proposed studies are designed to address the mechanisms through which cyclin D1 contributes to breast cancer using the pathophysiologically relevant experimental context of the intact animal. The studies will (a) examine whether overexpressed cyclin D1 may contribute to mammary tumorigenesis through mechanisms apart from activation of cdk4, by developing and characterizing transgenic mice with a mammary-targeted mutant cyclin D1 transgene unable to activate cdk4; (b) examine whether overexpressed cyclin D1 may contribute to mammary tumorigenesis through mechanisms that do not rely on its ability to interact with pRB, by developing and characterizing transgenic mice with a mammary-targeted mutant cyclin D1 transgene unable to interact with pRB; (c) examine whether overexpressed cyclin D1 may significantly contribute to mammary tumorigenesis through direct activation of the estrogen receptor, by developing and characterizing transgenic mice with a mammary-targeted mutant cyclin D1 transgene unable to activate the estrogen receptor. These results will carry important implications to the molecular pathogenesis of breast cancer, and may ultimately contribute to novel treatment strategies.